Analysis of gene mutations in exhaled breath condensate from healthy and lung cancer individuals and profiling of mutations and gut microbiota in stools from patients with gastrointestinal neoplasms

University of Helsinki, Faculty of Medicine, Medicum, Department of Pathology

en

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Biolääketieteellinen tohtoriohjelma

fi

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Doktorandprogrammet i biomedicin

sv

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Doctoral Programme in Biomedicin

en

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Youssef, Omar

dc.date.accessioned

2018-10-22T10:05:18Z

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2018-11-06

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2018-10-22T10:05:18Z

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2018-11-16

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URN:ISBN:978-951-51-4645-8

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http://hdl.handle.net/10138/252441

dc.description.abstract

Lung and gastrointestinal (GIT) cancers are two types of malignancies in which early diagnosis has a significant impact on prognosis and better survival rate. Exhaled breath condensate (EBC) from lung cancer patients and stool from patients with GIT tumors can represent non-invasive sources for diagnosis of malignancy at an early stage. These materials contain DNA from cells exfoliated from malignant or pre-malignant lesions and consequently could reflect all genetic alterations occurring during development of the cancer. Stool samples are also a good source to study gut bacterial composition. Changes in the gut bacterial profile are linked to many diseases including GIT cancers. The aim of the study was to explore gene mutations in these samples, and to test their feasibility for the detection of malignancy in different tumor stages, including both early and advanced stages. A further aim was to investigate differences in the gut microbiota profile in stool samples of GIT cancer patients based on the location of the tumor.
The study material consisted of EBC samples from 26 lung cancer patients and 20 healthy individuals and stool specimens collected from 87 GIT neoplasia patients and 14 healthy individuals included as controls. DNA was isolated from both the EBC and stool samples. Targeted amplicon next generation sequencing (NGS) and 16S rRNA sequencing, using the Ion Torrent platforms, were performed to study gene mutations and stool bacterial profiling, respectively.
In study I, the methodology was optimized for applying NGS to study gene mutations in the EBC DNA from healthy individuals. The results revealed 15 subjects showing a total of 35 hotspot mutations in their EBC samples. The most frequent hotspot mutations occurred at TP53, KRAS, NRAS, and SMAD4 genes. A codon 12 KRAS G12V mutation was detected in one control EBC sample with a mutant allele fraction of 6.8%. In the follow-up, study II, the same methodological steps were applied to the DNA isolated from EBC samples of patients with lung neoplasms. The success rate was 67.9% with 17 patients revealing a total of 39 hotspot mutations in their EBC. The most frequent hotspot mutations occurred in the following genes: TP53, SMAD4, PIK3CA, and KRAS. A codon 13 KRAS G13D mutation was detected in one patient’s EBC sample with a mutant allele fraction of 17%. The average mutant allele fraction for the gene mutations seen in patients were higher compared to that in controls; e.g. for TP53, the average mutant allele fraction was 22.9% and 13.6% and for KRAS, 11.4% and 4.3% in the patients and controls, respectively.
In study III, a cancer hotspot gene panel together with colon and lung cancer gene panels were used to study mutations in stool DNA from 87 patients with gastric and colorectal neoplasms. The success rates were 78% and 87% for gastric and colorectal neoplasia, respectively. Stools from patients with gastric neoplasms revealed 5 hotspot mutations, while from colorectal neoplasms 20 hotspot mutations were found. APC, TP53, and KRAS were the most frequently mutated genes in colorectal neoplasms. However, APC, CDKN2A, and EGFR were the only genes that showed hotspot mutations in gastric neoplasms. Hotspot mutations could also be detected in stool DNA from benign (8 mutations) and early malignant (9 mutations) GIT neoplasms.
In study IV, bacterial profiling in stool samples from patients with GIT neoplasms revealed variations in abundance according to the site of the GIT neoplasm. Two families, Lactobacillaceae and Bifidobacteriaceae, showed lower relative abundance while Enterobacteriaceae showed higher relative abundance when compared with control samples. The observed bacterial diversity could serve as an indicator in GIT neoplasms and help in disease monitoring.
To conclude, EBC and stool specimens are easily accessible non-invasive samples that could be used for studying different genetic alterations in neoplasms. Our studies revealed that NGS is a sensitive molecular technique that can be successfully applied to study gene mutations in multiple cancer genes from a very small amount of input DNA.